Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.

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Background-Rho-associated kinase (ROCK), an effector of small GTPase Rho, regulates vascular tone via a calcium sensitization mechanism and plays a key role in the pathogenesis of hypertension. However, its role in vascular growth remains unclear. Methods and Results-Y-27632, a specific ROCK inhibitor, and the overexpression of dominant-negative ROCK suppressed the mitogen-induced DNA synthesis of cultured vascular smooth muscle cells (VSMCs), which indicates the essential role of ROCK in the control of VSMC proliferation in vitro. Y-27632 also suppressed the chemotaxis of VSMCs. Male Wistar rats were systemically given Y-27632 (35 to 70 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) through an intraperitoneal infusion. The neointimal formation of balloon-injured carotid arteries was significantly suppressed in Y-27632-treated rats (intima/media ratio, 0.22Ϯ0.02) compared with vehicle-treated rats (intima/media ratio, 0.92Ϯ0.21) or hydralazine-treated rats with a similar blood pressure decrease (intima/media ratio, 1.03Ϯ0.15). The phosphorylation of myosin phosphatase and myosin light chain was elevated in injured arteries in a Y-27632-sensitive manner, indicating the augmentation of ROCK activity in neointimal formation. The downregulation of the cyclin-dependent kinase inhibitor p27 kip1 in injured vessels was reversed by Y-27632 treatment, reflecting the antiproliferative effect of ROCK inhibition in vivo. Conclusions-We conclude that ROCK plays a key role in the process of neointimal formation after balloon injury. Thus, the inhibition of ROCK may be a potential therapeutic strategy for treating vascular proliferative disorders and hypertension. Key Words: atherosclerosis Ⅲ muscle, smooth Ⅲ remodeling Ⅲ signal transduction Ⅲ hypertension E levated vascular tone contributes to the pathogenesis of hypertension. Rho-associated kinase (ROCK), 1 a target of small GTPase Rho, regulates vascular contractility by increasing the level of phosphorylated myosin light chain and thereby elevating the calcium sensitivity of vascular smooth muscle cells (VSMCs). 2 Recently, Uehata et al 3 developed a potent, specific, ROCK inhibitor, Y-27632. The administration of Y-27632 to several hypertensive rat models markedly reduced systolic blood pressure (SBP), implicating ROCK as a key mediator in the pathogenesis of hypertension. 3 We and others have reported that regulators of vascular tone, such as angiotensin II or natriuretic peptides, are also involved in vascular growth. 4 Thus, we postulated that intracellular mechanism(s) should exist that govern both vascular contraction and growth. Using Y-27632 and dominant-negative ROCK, the present study demonstrates that ROCK, the key regulator of vascular contraction, also controls vascular growth in vitro and in vivo. Methods MaterialsY-27632 was obtained from Yoshitomi Pharmaceutical Industries, Osaka, Japan. The pCAG-myc and pCAG-myc-KD-IA plasmids 1 were a gift from T. Ishizaki and S. Narumiya (Kyoto University). The pEXV-myc-N19RhoA was from M. Symons (the Picower Institute for Medical Research), and...

Lipid raft microdomains act as organizing centers for signal transduction. We report here that the exocyst complex, consisting of Exo70, Sec6, and Sec8, regulates the compartmentalization of Glut4-containing vesicles at lipid raft domains in adipocytes. Exo70 is recruited by the G protein TC10 after activation by insulin and brings with it Sec6 and Sec8. Knockdowns of these proteins block insulin-stimulated glucose uptake. Moreover, their targeting to lipid rafts is required for glucose uptake and Glut4 docking at the plasma membrane. The assembly of this complex also requires the PDZ domain protein SAP97, a member of the MAGUKs family, which binds to Sec8 upon its translocation to the lipid raft. Exocyst assembly at lipid rafts sets up targeting sites for Glut4 vesicles, which transiently associate with these microdomains upon stimulation of cells with insulin. These results suggest that the TC10/exocyst complex/SAP97 axis plays an important role in the tethering of Glut4 vesicles to the plasma membrane in adipocytes. INTRODUCTIONInsulin stimulates glucose transport in fat and muscle cells through a process of regulated vesicle recycling in which the facilitative glucose transporter Glut4 is translocated from intracellular sites to the plasma membrane (Saltiel and Kahn, 2001;Bryant et al., 2002). In unstimulated cells, Glut4 undergoes endocytosis into endosomes and subsequently sorts into specialized storage vesicles that traffic to the plasma membrane after activation of the insulin receptor. The vesicles then dock and fuse at specific sites at the membrane, resulting in extracellular exposure of the transporter. The precise signals from the insulin receptor that control these events involve the tyrosine phosphorylation of a number of intracellular substrates. These phosphorylations lead to activation of the PI3-kinase pathway (Saltiel and Kahn, 2001) and also to activation of the G protein TC10 Maffucci et al., 2003), which in turn binds to numerous effectors, including the exocyst protein Exo70 (Inoue et al., 2003). Exo70 exists in a multiprotein complex with the proteins Sec6 and Sec8. A dominant-negative mutant of Exo70 blocked insulin-stimulated glucose uptake, but was without effect on translocation of Glut4 to the plasma membrane. However, this Exo70 mutant prevented the appearance of Glut4 at the cell surface, leading us to propose that the exocyst complex may play a critical role in tethering Glut4 vesicles to the plasma membrane for subsequent docking and fusion (Inoue et al., 2003).Lipid rafts are specialized compartments of the plasma membrane enriched in cholesterol and glycosphingolipids. Numerous signaling and cytoskeletal proteins are found in these subdomains, suggesting that they may act as organizing centers for signal transduction, particularly for insulin (Anderson, 1998;Schlegel et al., 1998;Bickel, 2002; Pessin, 2002, 2003). Both the insulin receptor and TC10 reside in lipid rafts (Yamamoto et al., 1998;Gustavsson et al., 1999;Nystrom et al., 1999;Kimura et al., 2002;Vainio et al., 2002)...

These results demonstrate distinct expression of VEGF and its receptors (flt-1 and Flk-1) in atherosclerotic lesions in human coronary arteries. Considering the multipotent actions of VEGF documented experimentally in vivo and in vitro, our findings suggest that VEGF may have some role in the progression of human coronary atherosclerosis, as well as in recanalization processes in obstructive coronary diseases.

Developing a human-on-a-chip by connecting multiple model organ systems would provide an intermediate screen for therapeutic efficacy and toxic side effects of drugs prior to conducting expensive clinical trials. However, correctly designing individual organs and scaling them relative to each other to make a functional microscale human analog is challenging, and a generalized approach has yet to be identified. In this work, we demonstrate the importance of rational design of both the individual organ and its relationship with other organs, using a simple two-compartment system simulating insulin-dependent glucose uptake in adipose tissues. We demonstrate that inter-organ scaling laws depend on both the number of cells, and on the spatial arrangement of those cells within the microfabricated construct. We then propose a simple and novel inter-organ ‘metabolically-supported functional scaling’ approach predicated on maintaining in vivo cellular basal metabolic rates, by limiting resources available to cells on the chip. This approach leverages findings from allometric scaling models in mammals that limited resources in vivo prompts cells to behave differently than in resource-rich in vitro cultures. Although applying scaling laws directly to tissues can result in systems that would be quite challenging to implement, engineering workarounds may be used to circumvent these scaling issues. Specific workarounds discussed include the limited oxygen carrying capacity of cell culture media when used as a blood substitute and the ability to engineer non-physiological structures to augment organ function, to create the transport-accessible, yet resource-limited environment necessary for cells to mimic in vivo functionality. Furthermore, designing the structure of individual tissues in each organ compartment may be a useful strategy to bypass scaling concerns at the inter-organ level.

Insulin stimulates glucose uptake by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly; the mechanism underlying this process remains uncertain. Using the TC10-interacting protein CIP4 as bait in a yeast two-hybrid screen, we cloned a RasGAP and VPS9 domain-containing protein, Gapex-5/RME-6. The VPS9 domain is a guanine nucleotide exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trans-Golgi network (TGN)-to-endosome trafficking. Overexpression of Rab31 blocks insulin-stimulated Glut4 translocation, whereas knockdown of Rab31 potentiates insulin-stimulated Glut4 translocation and glucose uptake. Gapex-5 is predominantly cytosolic in untreated cells; its overexpression promotes intracellular retention of Glut4 in adipocytes. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, thus reducing Rab31 activity and permitting Glut4 vesicles to translocate to the cell surface, where Glut4 docks and fuses to transport glucose into the cell.

Abstract-Vascular endothelial growth factor